The field of this invention is the area of bacterial genetics, lipooligosaccharide biosynthesis, vaccines and lipid A-containing compositions, and in particular, as related to Neisseria meningitidis. 
Neisseria meningitidis and Neisseria gonorrhoeae are important human pathogens. N. meningitidis causes meningitis, sepsis and bacteremia; N. gonorrhoeae causes gonorrhea in both sexes, pelvic inflammatory disease and/or sterility in women, and rectal and pharyngeal infections, as in homosexual men. More rarely, disseminated gonococcal infection (gonococcal bacteremia) can result, with complications such as polyarthralgias or purulent arthritis, for example. These two species are relatively closely related genetically; there is approximately 85% DNA sequence homology between the genomes of the two species. The genus also includes several other species which are nonpathogenic to man although they colonize the upper respiratory tract.
Neisseria produce lipooligosaccharide (LOS) which is associated with the bacterial outer membrane. The lipooligosaccharide differs from the lipopolysaccharide (LPS) of the Enterobacteriaceae in that they are short, often branched sugar chains rather than relatively long repeating subunits. Neisserial LOS is classified into six serotypes among the gonococci and into thirteen in the meningococci. Neisserial LOS contain glucose, galactose, 2-keto-3-deoxyoctonic acid (Kdo), glucosamine, galactosamine, sialic acid and ethanolamine in ratios and linkages which depend on the serotype. LOS molecules produced by wild-type strains generally have molecular masses in the range of about 3200 to about 7000 d, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The short, often branched oligosaccharide chains are attached via Kdo to lipid A embedded in the outer membrane. The LOS structure of a particular strain is subject to antigenic variation.
Lipid A of the neisseriae acts as a classic endotoxin and can induce changes in the permeability of the blood brain barrier after invasion of the cerebrospinal fluid during meningococcemia (Tunkel and Scheld (1993) Clin. Res. Microbiol. 6, 118-136). The composition of the LOS influences the invasive capacity of the meningococci (MacKinnon et al. (1993) Microb. Path. 15, 359-366) and in the gonococci as well as the meningococci, the composition of the LOS affects the susceptibility of the bacterial cells to normal human serum (Shafer et al. (1984) J. Infec. Dis. 149, 179-183; Porat et al. (1995) Infect. Immun. 63:2164-2172).
The morbidity and mortality of meningococcal bacteremia and meningitis have been directly correlated with the amount of circulating meningococcal endotoxin (lipopoly[oligo]saccharide or LOS) (van Deuren, 2000; Brandtzaeg, 1989; Brandtzaeg, 1992). The engagement of meningococcal LOS with the human toll-like receptor 4 (TLR4) on human macrophages and other host cells is proposed to trigger signaling events that ultimately result in cytokine gene activation and the production of proinflammatory cytokines and chemokines. Meningococcemia and meningococcal meningitis are predicted in large part to be a direct result of the over-stimulation of TLR4 activation by circulating meningococcal LOS (Brandtzaeg, 1989; Brandtzaeg, 1992; Brandtzaeg, 1995) inducing a cascade of events that lead clinically to hypotension, organ failure, necrosis, coma and death. However, the mechanism by which meningococcal LOS activates TLR4 to produce fulminant meningococcemia and meningitis is not understood.
Neisseria meningitidis, an exclusive human pathogen, is a cause of bacterial meningitis and sepsis, and infection can result in epidemic as well as endemic disease. Capsular polysaccharides and lipooligosaccharide (LOS) are two critical virulence factors in meningococcal pathogenesis (Tzeng and Stephens, 2000), contributing to the resistance of meningococci to serum bactericidal activity (Kahler et al., 1998). Capsular polysaccharides protect meningococci from host immune defenses, including phagocytosis, opsonization and complement-mediated killing (Jarvis, 1995; Troy, 1992). Capsule also protects meningococci from environmental stress such as desiccation and facilitates transmission due to its anti-adherence properties (Stephens and McGee, 1981; Stephens et al., 1993; Virji et al, 1993). Mimicry by LOS structure of the carbohydrate moieties of glycosphingolipids present in many human cells (Estabrook et al., 1997; Moran et al., 1996) further enables meningococci to escape bactericidal antibody recognition.
Structural differences in capsule and LOS are the determinants in the serological typing of meningococcal serogroups and immunotypes respectively. Of the thirteen different capsule serogroups so far defined, five (serogroups A, B, C, Y, and W-135) are associated with invasive meningococcal disease. Serogroup A capsule is (α1→6) linked N-acetyl mannosamine 1-phosphate; serogroup B capsule is composed of (α2→8) linked N-acetylneuraminic acid (NANA); serogroup C capsule is (α2→9) linked partially O-acetylated NANA; serogroup Y capsule is an alternating sequence of D-glucose and partially O-acetylated NANA; and serogroup W-135 capsule is composed of alternating sequence of D-galactose and NANA. Meningococcal LOS consists of lipid A, a conserved inner core composed of two heptoses linked to two 3-deoxy-D-manno-2-octulosonic acid moieties (Kdo), and an outer core with variable oligosaccharide composition. The meningococcal lipid A is distinct from that of E. coli; it is composed of a β1′, 6-linked disaccharide of glucosamine acetylated with β-hydroxymyristates and β-hydroxylaurates at the 2, 2′ and 3, 3′ positions, respectively, and symmetrical acyloxyacyl linkages of laurate residues are located at the 2, 2′ positions (Rahman et al., 1998).
More than thirty genes involved in the biosynthesis of lipid A, heptose, Kdo and the outer core polysaccharides have been identified (Kahler and Stephens, 1998) The capsule biosynthetic pathway has also been studied extensively. A four-gene operon (synABCD) mediates the production of sialic acid and the formation of capsule polymers; while the divergently transcribed ctrABCD operon encodes the proteins responsible for capsule translocation (Swartley et al., 1996). No genes outside the capsule locus have been shown, as yet, to participate in capsule expression.
E. coli K1 strains also express a capsule composed of (α2→8) linked polysialic acid. The capsule locus of K1 E. coli has also been well characterized, and when compared to the meningococcal capsule locus (FIG. 1A), it contains several “extra” genes including kpsF, kpsD, kpsU, neuD and neuE. KpsU has been shown to encode a second copy of the CMP-Kdo synthetase, KdsB (Rosenow et al., 1995). KpsD is a periplasmic protein, and mutation of kpsD resulted in periplasmic polysaccharide. The functions of KpsF, NeuD and NeuE are currently unknown. Prior to the present invention, it was not known if these genes were present in meningococci.
There is a long felt need in the art for a protective vaccine effective in the prevention of human diseases caused by the pathogenic Neisseria species, N. gonorrhoeae and N. meningitidis, especially Group B meningococci. Meningococcal meningitis or meningococcemia can have about 85% mortality if untreated and about 10-20% if treated, and individuals with deficiencies in late complement cascade components C5, C6, C7 and C8 appear to be prone to multiple episodes of meningococcal meningitidis. For example, nonpathogenic strains or antigenic material therefrom, particularly those which lack intact lipooligosaccharide (LOS) structure, as antigen for preparing antibodies specific to this bacterial surface component or for attenuated vaccines useful in protection against the diseases resulting from infection with Neisseria species. There is also a need in the art for Lipid A-producing strains of bacteria, where the purification and preparation of lipid A is simplified in comparison to preparation from enteric bacteria or neisseriae with intact LOS.